Hypertrophic Alveolar Type II Cells from Silica-Treated Rats are Committed to DNA Synthesis In Vitro

Alveolar type II cell hyperplasia and hypertrophy are common reparative responses of the alveolar epithelium after silica-induced lung injury. We studied in vitro DNA synthesis in type II cells isolated after silica instillation in the rat to determine the proliferative potential of silica type II cells in primary culture and to correlate alveolar type II cell size with the level of in vitro DNA synthesis. To determine if the alveolar lining fluid is a source of growth factors that stimulate alveolar type II cell proliferation, we also examined the mitogenic effect of bronchoalveolar lavage fluid (BALF) from silica-treated rats on type II cells in primary culture. Alveolar type II cells were isolated from rats 1, 2, 3, and 4 wk after intratracheal silica instillation, cultured in DME supplemented with 10% fetal bovine serum, and labeled with [3H]thymidine from day 1 to day 3 in culture. DNA synthesis was determined by [3H]thymidine incorporation and autoradiographic labeling index. The level of thymidine incorporation increased progressively from 22.3 ± 5.4 × 103 dpm/well 7 d after silica instillation to 34.4 ± 5.0 × 103 dpm/well at 28 d. Type II cells isolated 14 d after silica instillation were separated into groups of increasing cell size by centrifugal elutriation. The plating efficiency and alveolar type II cell purity (> 88%) were the same in all groups of elutriated cells. The hypertrophic type II cells had a higher level of thymidine incorporation (22.0 ± 2.8 × 103 dpm/well) than the normotrophic type II cells (11.1 ± 0.7 × 103 dpm/well) [P < 0.01]). Each group of elutriated type II cells was capable of responding to the same level of stimulated DNA synthesis in the presence of insulin, epidermal growth factor, and cholera toxin. The autoradiographic labeling index increased from 6.2 ± 0.8% in the normotrophic type II cells to 15.8 ± 2.1% in the hypertrophic cells compared to 1.5 ± 0.01% in type II cells isolated from saline control animals. BALF from silica-treated rats stimulated the same level of DNA synthesis as that induced by BALF from untreated animals. We conclude that hypertrophic type II cells are a subgroup of alveolar type II cells that are committed to DNA synthesis in primary culture and that the mitogenic factor(s) for alveolar type II cells found in BALF from normal rats is not increased after silica instillation.